Co-enrichment of cancer-associated bacterial taxa is correlated with immune cell infiltrates in esophageal tumor tissue.

Esophageal carcinoma (ESCA) is a leading cause of cancer-related death worldwide, and certain oral and intestinal pathogens have been associated with cancer development and progression. We asked if esophageal microbiomes had shared alterations that could provide novel biomarkers for ESCA risk. We extracted DNA from tumor and non-tumor tissue of 212 patients in the NCI-MD case control study and sequenced the 16S rRNA gene (V3-4), with TCGA ESCA RNA-seq (n = 172) and WGS (n = 123) non-human reads used as validation. We identified four taxa, Campylobacter, Prevotella, Streptococcus, and Fusobacterium as highly enriched in esophageal cancer across all cohorts. Using SparCC, we discovered that Fusobacterium and Prevotella were also co-enriched across all cohorts. We then analyzed immune cell infiltration to determine if these dysbiotic taxa were associated with immune signatures. Using xCell to obtain predicted immune infiltrates, we identified a depletion of megakaryocyte-erythroid progenitor (MEP) cells in tumors with presence of any of the four taxa, along with enrichment of platelets in tumors with Campylobactor or Fusobacterium. Taken together, our results suggest that intratumoral presence of these co-occurring bacterial genera may confer tumor promoting immune alterations that allow disease progression in esophageal cancer.

Hormone use and risk for lung cancer: a pooled analysis from the International Lung Cancer Consortium (ILCCO).

BACKGROUND: The association between oral contraceptive (OC) use, hormone replacement therapy (HRT) and lung cancer risk in women is still debated. METHODS: We performed a pooled analysis of six case-control studies (1961 cases and 2609 controls) contributing to the International Lung Cancer Consortium. Potential associations were investigated with multivariable unconditional logistic regression and meta-analytic models. Multinomial logistic regressions were performed to investigate lung cancer risk across histologic types. RESULTS: A reduced lung cancer risk was found for OC (odds ratio (OR)=0.81; 95% confidence interval (CI): 0.68-0.97) and HRT ever users (OR=0.77; 95% CI: 0.66-0.90). Both oestrogen only and oestrogen+progestin HRT were associated with decreased risk (OR=0.76; 95% CI: 0.61-0.94, and OR=0.66; 95% CI: 0.49-0.88, respectively). No dose-response relationship was observed with years of OC/HRT use. The greatest risk reduction was seen for squamous cell carcinoma (OR=0.53; 95% CI: 0.37-0.76) in OC users and in both adenocarcinoma (OR=0.79; 95% CI: 0.66-0.95) and small cell carcinoma (OR=0.37; 95% CI: 0.19-0.71) in HRT users. No interaction with smoking status or BMI was observed. CONCLUSION: Our findings suggest that exogenous hormones can play a protective role in lung cancer aetiology. However, given inconsistencies with epidemiological evidence from cohort studies, further and larger investigations are needed for a more comprehensive view of lung cancer development in women.

Genetic interactions between tumor suppressors Brca1 and p53 in apoptosis, cell cycle and tumorigenesis.

Breast cancer is a chief cause of cancer-related mortality that affects women worldwide. About 8% of cases are hereditary, and approximately half of these are associated with germline mutations of the breast tumor suppressor gene BRCA1 (refs. 1,2). We have previously reported a mouse model in which Brca1 exon 11 is eliminated in mammary epithelial cells through Cre-mediated excision. This mutation is often accompanied by alterations in transformation-related protein 53 (Trp53, encoding p53), which substantially accelerates mammary tumor formation. Here, we sought to elucidate the underlying mechanism(s) using mice deficient in the Brca1 exon 11 isoform (Brca1Delta11/Delta11). Brca1Delta11/Delta11 embryos died late in gestation because of widespread apoptosis. Unexpectedly, elimination of one Trp53 allele completely rescues this embryonic lethality and restores normal mammary gland development. However, most female Brca1Delta11/Delta11 Trp53+/- mice develop mammary tumors with loss of the remaining Trp53 allele within 6-12 months. Lymphoma and ovarian tumors also occur at lower frequencies. Heterozygous mutation of Trp53 decreases p53 and results in attenuated apoptosis and G1-S checkpoint control, allowing Brca1Delta11/Delta11 cells to proliferate. The p53 protein regulates Brca1 transcription both in vitro and in vivo, and Brca1 participates in p53 accumulation after gamma-irradiation through regulation of its phosphorylation and Mdm2 expression. These findings provide a mechanism for BRCA1-associated breast carcinogenesis.

SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.

Hepatocellular carcinoma (HCC) is a major cause of cancer death, but the molecular mechanism for its development beyond its initiation has not been well characterized. Suppressor of cytokine signaling (SOCS-1; also known as JAB and SSI-1) switches cytokine signaling 'off' by means of its direct interaction with Janus kinase (JAK). We identified aberrant methylation in the CpG island of SOCS-1 that correlated with its transcription silencing in HCC cell lines. The incidence of aberrant methylation was 65% in the 26 human primary HCC tumor samples analyzed. Moreover, the restoration of SOCS-1 suppressed both growth rate and anchorage-independent growth of cells in which SOCS-1 was methylation-silenced and JAK2 was constitutively activated. This growth suppression was caused by apoptosis and was reproduced by AG490, a specific, chemical JAK2 inhibitor that reversed constitutive phosphorylation of STAT3 in SOCS-1 inactivated cells. The high prevalence of the aberrant SOCS-1 methylation and its growth suppression activity demonstrated the importance of the constitutive activation of the JAK/STAT pathway in the development of HCC. Our results also indicate therapeutic strategies for the treatment of HCC including use of SOCS-1 in gene therapy and inhibition of JAK2 by small molecules, such as AG490.

p53 and vascular endothelial growth factor regulate tumor growth of NOS2-expressing human carcinoma cells.

The finding of frequent nitric oxide synthase expression in human cancers indicates that nitric oxide has a pathophysiological role in carcinogenesis. To determine the role of nitric oxide in tumor progression, we generated human carcinoma cell lines that produced nitric oxide constitutively. Cancer cells expressing inducible nitric oxide synthase that had wild-type p53 had reduced tumor growth in athymic nude mice, whereas those with mutated p53 had accelerated tumor growth associated with increased vascular endothelial growth factor expression and neovascularization. Our data indicate that tumor-associated nitric oxide production may promote cancer progression by providing a selective growth advantage to tumor cells with mutant p53, and that inhibitors of inducible nitric oxide synthase may have therapeutic activity in these tumors.

Distribution and modulation of the cellular receptor for transforming growth factor-beta.

Scatchard analyses of the binding of transforming growth factor-beta (TGF-beta) to a wide variety of different cell types in culture revealed the universal presence of high affinity (Kd = 1-60 pM) receptors for TGF-beta on every cell type assayed, indicating a wide potential target range for TGF-beta action. There was a strong (r = +0.85) inverse relationship between the receptor affinity and the number of receptors expressed per cell, such that at low TGF-beta concentrations, essentially all cells bound a similar number of TGF-beta molecules per cell. The binding of TGF-beta to various cell types was not altered by many agents that affect the cellular response to TGF-beta, suggesting that modulation of TGF-beta binding to its receptor may not be a primary control mechanism in TGF-beta action. Similarly, in vitro transformation resulted in only relatively small changes in the cellular binding of TGF-beta, and for those cell types that exhibited ligand-induced down-regulation of the receptor, down-regulation was not extensive. Thus the strong conservation of binding observed between cell types is also seen within a given cell type under a variety of conditions, and receptor expression appears to be essentially constitutive. Finally, the biologically inactive form of TGF-beta, which constitutes greater than 98% of autocrine TGF-beta secreted by all of the twelve different cell types assayed, was shown to be unable to bind to the receptor without prior activation in vitro. It is proposed that this may prevent premature interaction of autocrine ligand and receptor in the Golgi apparatus.

p53 mutations in human cancers.

Mutations in the evolutionarily conserved codons of the p53 tumor suppressor gene are common in diverse types of human cancer. The p53 mutational spectrum differs among cancers of the colon, lung, esophagus, breast, liver, brain, reticuloendothelial tissues, and hemopoietic tissues. Analysis of these mutations can provide clues to the etiology of these diverse tumors and to the function of specific regions of p53. Transitions predominate in colon, brain, and lymphoid malignancies, whereas G:C to T:A transversions are the most frequent substitutions observed in cancers of the lung and liver. Mutations at A:T base pairs are seen more frequently in esophageal carcinomas than in other solid tumors. Most transitions in colorectal carcinomas, brain tumors, leukemias, and lymphomas are at CpG dinucleotide mutational hot spots. G to T transversions in lung, breast, and esophageal carcinomas are dispersed among numerous codons. In liver tumors in persons from geographic areas in which both aflatoxin B1 and hepatitis B virus are cancer risk factors, most mutations are at one nucleotide pair of codon 249. These differences may reflect the etiological contributions of both exogenous and endogenous factors to human carcinogenesis.

Genotoxicity of formaldehyde in cultured human bronchial fibroblasts.

Formaldehyde, a common environmental pollutant, inhibits repair of O6-methylguanine and potentiates the mutagenicity of an alkylating agent, N-methyl-N-nitrosourea, in normal human fibroblasts. Because formaldehyde alone also causes mutations in human cells, the compound may cause genotoxicity by a dual mechanism of directly damaging DNA and inhibiting repair of mutagenic and carcinogenic DNA lesions caused by other chemical and physical carcinogens.

Geographic variation of p53 mutational profile in nonmalignant human liver.

Fifty-eight percent of hepatocellular carcinomas (HCCs) from Qidong, China, contain an AGG to AGT mutation at codon 249 of the p53 tumor suppressor gene, a mutation that is rarely seen in HCCs from Western countries. The population of Qidong is exposed to high levels of aflatoxin B1 (AFB1), a fungal toxin that has been shown to induce the same mutation in cultured human HCC cells. To investigate the role of AFB1 and of these p53 mutations in hepatocarcinogenesis, normal liver samples from the United States, Thailand, and Qidong (where AFB1 exposures are negligible, low and high, respectively) were examined for p53 mutations. The frequency of the AGG to AGT mutation at codon 249 paralleled the level of AFB1 exposure, which supports the hypothesis that this toxin has a causative--and probably early--role in hepatocarcinogenesis.

Formaldehyde damage to DNA and inhibition of DNA repair in human bronchial cells.

Cultured bronchial epithelial and fibroblastic cells from humans were used to study DNA damage and toxicity caused by formaldehyde. Formaldehyde caused the formation of cross-links between DNA and proteins, caused single-strand breaks in DNA, and inhibited the resealing of single-strand breaks produced by ionizing radiation. Formaldehyde also inhibited the unscheduled DNA synthesis that occurs after exposure of cells to ultraviolet irradiation or to benzo[a]pyrene diolexpoxide but at doses substantially higher than those required to inhibit the resealing of x-ray-induced single-strand breaks. Therefore, formaldehyde could exert its mutagenic and carcinogenic effects by both damaging DNA and inhibiting DNA repair.

Interindividual variation in binding of benzo[a]pyrene to DNA in cultured human bronchi.

The binding of benzo[a]pyrene to DNA in cultured human bronchus was measured in specimens from 37 patients. The binding values ranged from 2 to 151 picomoles of benzo[a]pyrene per milligram of DNA with an overall mean +/- standard error of 34.2 +/- 5.2. This 75-fold interindividual variation in the binding of benzo[a]pyrene to DNA is similar in magnitude to that found in pharmacogenetic studies of drug metabolism. Aryl hydrocarbon hydroxylase is also inducible by benz[a]anthracene in the bronchial mucosa.

RNA metabolism in tracheal epithelium: alteration in hamsters deficient in vitamin A.

The electrophoretic pattern of RNA molecules that are synthesized in vitro in tracheal epithelium from hamsters deficient in vitamin A differs from that of RNA synthesized in normal, pair-fed control hamsters. There is less RNA of low electrophoretic mobility in the epithelial cells deficient in vitamin A. This alteration is reversed after the deficient animals have been treated with vitamin A.

Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene.

Transfection of normal human bronchial epithelial (NHBE) cells with a plasmid carrying the ras oncogene of Harvey murine sarcoma virus (v-Ha ras) changed the growth requirements, terminal differentiation, and tumorigenicity of the recipient cells. One of the cell lines isolated after transfection (TBE-1) was studied extensively and shown to contain v-Ha ras DNA. Total cellular RNA from TBE-1 cells hybridized to v-Ha ras structural gene fragment probes five to eight times more than RNA from parental NHBE cells. The TBE-1 cells expressed phosphorylated v-Ha ras polypeptide p21, showed a reduced requirement for growth-factor supplements, and became aneuploid as an early cellular response to v-Ha ras expression. As the transfectants acquire an indefinite life-span and anchorage independence they became transplantable tumor cells and showed many phenotypic changes suggesting a pleiotropic mechanism for the role of Ha ras in human carcinogenesis.

High-frequency transfection and cytopathology of the hepatitis B virus core antigen gene in human cells.

A protoplast fusion method was developed to stably transfect human cells with pSV2-derived plasmids at frequencies greater than 10(-3). This procedure made it possible to test the biological effect of a hepatitis B virus (HBV) gene independent of the viral structures required for infection. A pSV2gpt+ plasmid constructed to carry a subgenomic fragment of HBV that contained the core antigen gene (HBc gene) was transfected into human cells. A human epithelial cell line was stably transfected with the HBc+ gene by selecting recipient cells for expression of guanine phosphoribosyl transferase expression. With this gpt+/HBc+ cell line it was shown that growth in serum-free medium or treatment with 5'-azacytidine stimulates the production of the HBV core antigen. A hepatocellular carcinoma carrying the entire HBV genome was stimulated to produce the HBc gene product in response to the same factors that stimulated HBcAg production in the gpt+/HBc+ cell line constructed by transfection. The temporal relation between the cytopathologic response and HBc gene expression was similar for both cell types, indicating a primary role for HBc gene expression in the cytopathology of HBV-infected human liver.

Paediatric intensive care in the field hospital.

Our recent experience of paediatric critical care during UK military operations in Afghanistan is discussed alongside consideration of the background to the paediatric critical care service on deployment. We describe the intensive care unit's capabilities, details of recent paediatric critical care admissions during July to September 2008 and some of the ethical issues arising. Some desirable future developments will be suggested.

TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the major risk factors include chronic infections with the hepatitis B (HBV) or C (HCV) virus, and exposure to dietary aflatoxin B(1) (AFB(1)) or alcohol consumption. Multiple genetic and epigenetic changes are involved in the molecular pathogenesis of HCC, for example, somatic mutations in the p53 tumor suppressor gene (TP53) and the activation of the WNT signal transduction pathway. AFB(1) frequently induces G:C to T:A transversions at the third base in codon 249 of TP53 and cooperates with HBV in causing p53 mutations in HCC. The detection of TP53 mutant DNA in plasma is a biomarker of both AFB(1) exposure and HCC risk. Chronic infection with HBV and HCV viruses, and oxyradical disorders including hemochromatosis, also generate reactive oxygen/nitrogen species that can both damage DNA and mutate cancer-related genes such as TP53. Certain mutant p53 proteins may exhibit a 'gain of oncogenic function'. The p53 biological network is a key responder to this oxidative and nitrosative stress. Depending on the extent of the DNA damage, p53 regulates the transcription of protective antioxidant genes and with extensive DNA damage, transactivates pro-oxidant genes that contribute to apoptosis. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC and the integrated HBx is frequently mutated. Mutant HBx proteins still retain their ability to bind to p53, and attenuate DNA repair and p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology of TP53 mutations during the molecular pathogenesis of HCC.

Bloom's syndrome workshop focuses on the functional specificities of RecQ helicases.

Human cells express five DNA helicases that are paralogs of Escherichia coli RecQ and which constitute the family of human RecQ helicases. Disease-causing mutations in three of these five human DNA helicases, BLM, WRN, and RECQL4, cause rare severe human genetic diseases with distinct clinical phenotypes characterized by developmental defects, skin abnormalities, genomic instability, and cancer susceptibility. Although biochemical and genetic evidence support roles for all five human RecQ helicases in DNA replication, DNA recombination, and the biological responses to DNA damage, many questions concerning the various functions of the human RecQ helicases remain unanswered. Researchers investigating human and non-human RecQ helicases held a workshop on May 27-28, 2008, at the University of Chicago Gleacher Center, during which they shared insights, discussed recent progress in understanding the biochemistry, biology, and genetics of the RecQ helicases, and developed research strategies that might lead to therapeutic approaches to the human diseases that result from mutations in RecQ helicase genes. Some workshop sessions were held jointly with members of a recently formed advocacy and support group for persons with Bloom's syndrome and their families. This report describes the outcomes and main discussion points of the workshop.

Redundancy of DNA helicases in p53-mediated apoptosis.

A subset of DNA helicases, the RecQ family, has been found to be associated with the p53-mediated apoptotic pathway and is involved in maintaining genomic integrity. This family contains the BLM and WRN helicases, in which germline mutations are responsible for Bloom and Werner syndromes, respectively. TFIIH DNA helicases, XPB and XPD, are also components in this apoptotic pathway. We hypothesized that there may be some redundancy between helicases in their ability to complement the attenuated p53-mediated apoptotic levels seen in cells from individuals with diseases associated with these defective helicase genes. The attenuated apoptotic phenotype in Bloom syndrome cells was rescued not only by ectopic expression of BLM, but also by WRN or XPB, both 3' --> 5' helicases, but not expression of the 5' --> 3' helicase XPD. Overexpression of Sgs1, a WRN/BLM yeast homolog, corrected the reduction in BS cells only, which is consistent with Sgs1 being evolutionarily most homologous to BLM. A restoration of apoptotic levels in cells from WS, XPB or XPD patients was attained only by overexpression of the specific helicase. Our data suggest a limited redundancy in the pathways of these RecQ helicases in p53-induced apoptosis.

Induction of ouabain-resistant mutation and sister chromatid exchanges in Chinese hamster cells with chemical carcinogens mediated by human pulmonary macrophages.

Pulmonary macrophages (PAM) metabolically activated benzo[a]pyrene [B(a)P] and its proximate carcinogenic metabolite, (+/-)trans 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol), to ultimate mutagens that were detected in cocultivated Chinese hamster V79 cells. Increases in the frequency of ouabainresistant (O(r)) mutations and sister chromatid exchanges were found in V79 cells only when they were cocultivated with both PAM and the chemical procarcinogens. 7,8-Diol caused higher frequencies of both O(r) mutations and sister chromatid exchanges than did the parent compound, B(a)P. When metabolically activated by PAM the mean O(r) mutation frequency caused by B(a)P was 9 O(r) mutants/10(6) surviving V79 cells per 10(6) PAM and a 10-fold interindividual variation (range, 2-21) was found. The mean O(r) mutation frequency caused by 7,8-diol was 64 and a ninefold interindividual variation (range, 14-120) was found. In the absence of PAM, the O(r) mutation frequency in V79 cells was one or less O(r) mutant per 10(6) survivors. 7,8-Benzoflavone, an inhibitor of mixed function oxidases, reduced the frequencies of O(r) mutations and of sister chromatid exchanges in V79 cells caused by 7,8-diol and B(a)P. As expected 7,8-benzoflavone did not influence the frequency of O(r) mutations caused by one of the ultimate mutagens derived from B(a)P and 7,8-diol, (+/-)7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. These data are consistant with the hypothesis that PAM may play a role in the activation of environmental chemical procarcinogens.

Detection and characterization of human serum antibodies to polycyclic aromatic hydrocarbon diol-epoxide DNA adducts.

The presence of serum antibodies to the diol-epoxide DNA adducts of representative polycyclic aromatic hydrocarbons (PAH), chrysene, benz[a]anthracene and benzo[a]pyrene, was determined by ELISA using serum samples obtained from normal healthy individuals. Antibodies that reacted against PAH adducted-DNA, but not against PAH-adducted protein, were found in the serum of approximately 40% of the test individuals. Specificity analysis of the antibodies demonstrated that serological cross-reactions between the benzo[a]pyrene and the chrysene diol-epoxide adducts were present. Similar cross-reactivity between the benz[a]anthracene and the chrysene adducts was observed. Sera containing antibodies that were apparently specific for each of the three PAH-DNA adducts were also identified. The presence of antibodies to PAH-DNA adducts indicates both past exposure to these carcinogenic PAH and their metabolic activation to the DNA damaging metabolites. These antibodies may prove to be useful in both retrospective and prospective epidemiological studies of various diseases associated with PAH exposure.